Emerging clinical data in the HLA-matched setting suggest that high-dose post-transplant cyclophosphamide (Cy) given as a single agent is effective in preventing both acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (alloHSCT). Patients who undergo transplants using this strategy have a low incidence of infections, and despite advanced disease stage, their survival rate is favorable, suggesting that this approach retains the graft-versus-leukemia effect. The exact mechanisms behind the efficacy of high-dose Cy as a post-transplant immunosuppressant are not fully understood, and systematic study of the correlates of T-cell immune recovery in the context of our ongoing multiinstitutional clinical trial will be crucial for complete understandingof the mechanisms. We hypothesize that high-dose Cy prevents acute GVHD by reducing the frequency of effector T cells while sparing the critical regulatory T-cell (Treg) pool. In addition we predict that the rapid establishment of regulatory T-cell homeostasis is critical for the development of tolerance and the absence of chronic GVHD. Finally, we hypothesize that grade 2-4 acute GVHD that requires treatment with conventional immune suppression after high-dose Cy prophylaxis will be associated with a significant delay in the recovery of a diverse T-cell receptor (TCR) repertoire in the Treg compartment. To test the validity of our hypotheses, we will first test for associations between the cellular and molecular changes in the effector and regulatory T-cell subsets and the development of grade 2-4 acute GVHD in patients treated with post-transplantation Cy. Second, we will determine whether the absence of chronic GVHD and the development of tolerance are influenced by Treg repertoire reconstitution and thymic recovery. Third, by measuring the TCR 2 chain CDR3 region sequence diversity in the naive, memory and regulatory T- cell compartments using high-throughput DNA sequencing technology, we will analyze whether any significant GVHD that occurs after the high-dose Cy prophylaxis is associated with the delayed reconstitution of a diverse Treg repertoire. The work proposed is expected to provide essential mechanistic insights supporting the use of a short course of GVHD prophylaxis that promotes tolerance induction and provides immune system reconstitution in an environment free of ongoing pharmacologic immunosuppression. It will also provide a high- resolution picture of the immune spectra in T-cell subsets and illuminate a path toward better control of alloreactivity.